Sterilizing liquids



Patented Oct. 26, -1948 STERILIZIN G LIQUIDS Sidney Older Levinson and Franz Oppenheimer, Chicago, Ill., asslgnors to Michael Reese Research Foundation, Chicago, Ill., a corporation f Illinois Application December 31, 1946, Serial No. '119,388

7 Claims.

This invention relates to the vsterilization o! liquid solutions or suspensions which are contaminated with micro-organisms without altering the chemical nature or impairing the biologic activity of substances in the solution. By means of th'e present invention we are able to produce sterile solutions or suspensions of delicate compounds which cannot be satisfactorily sterilized by the now commony practiced methods of sterilization.

At present, the accepted methods of sterilization of solutions or suspensions are:

(1) Steam sterilization under pressure for twenty to thirty minutes, or'dry heat sterilization at 180 C. for two hours.

(2) The addition of bactericidal chemicals.

(3) Filtration through filters of a selected porosity to remove certaintypes of micro-organisms.

None of the three methods is completely satisfactory for sterilizing liquids containing delicate substances. Heat will coagulate proteins or otherwise change unstable compounds. Chemicals may also adversely affect such delicate compounds. Upon standing chemicals will progressively act on delicate compounds and the potency or chemical activity of these compounds may be progressively weakened. Furthermore, the presence of these chemicals in the solutions is often dangerous and harmful if the solutions are to be used for humans. The filtration process has two disadvantages; rst, illterable viruses will not be removed, and secondly, there is a loss of the active material by adsorption to the filter.

The present invention comprises a process whereby contaminated solutions or suspensions can be completely sterilized and all viruses inactivated without materially impairing the potency or changing the chemical cate compounds contained in thel solution. This result is accomplished by the irradiation of flowing thin films of such solutions or suspensions for. a few seconds with rays emitted by an extremelow-pressure mercury arc discharge. The lamp which we employ is described in the copending application of Franz Oppenheimer, Serial No` 581,405, filed March 7, 1945. This extreme-lowpressure arc discharge emits radiation in powerful intensities in the region between 1800 and 2600 Prior eilorts to sterilize contaminated suspen sions containing delicate compounds with ultraviolet light have not produced satisfactory results. In these attempts, so called cold quartz lamps or ordinary quartz mercury arcs were employed. Relatively long exposures, from several minutes to one hour, were necessary to completely sterilize such solutions. These long exposures necessarily heat up the solutions, and have a deleterious effect upon delicate substances.

The lamps used, such as the cold-quartz lamp or the hot-cathode mercury discharge lamp, do not activity of any delisolutions with a .l G3) The ultraviolet (Cl. Z50-43) produce short ultra-violet radiation in great intensities. The lamp described in the copending application, in the range from 1800 to 2600 A., in intensities from 10 to 100 milliwatts per square centimeter. These measurements have been made by exposing one centimeter of the radiation tube at a distance of one centimeter from the lamp, Under similar` conditions, lamps of the coldor hot-cathode discharge type produce not more than one milliwatt of radiation, of which eighty-five percent is concentrated in of the lamps heretofore used for sterilization has any appreciable amount of radiation below 2000 while the lamp which we employ produces approximately twenty to thirty percent oi its energy in the ultraviolet in radiation below 2000 The time necessary to sterilize contaminated suspensions or solutions with lamps previously known is so long that the resultant products are unsatisfactory. Many investigators concluded that, while it takes a certain amount of ultraviolet energy to sterilize bacteria and inactive viruses, the time inwhich this energy is given to the suspension of micro-organism is of little consequence. Our investigations have shown that thereis a minimum amount of ultraviolet energy to which a solution containing a certain number of bacteria or viruses must be exposed in a certain time unit to render it sterile, and it is not possible to achieve the same result by irradiating these like total amount of ultraviolet energy in a correspondingly longer time. It has been determined that a minimum of 5 to 10 milliwatt seconds per square centimeter of energy of the wavelength 2537 is required to produce adequate killing of micro-organisms (Acceptance of ultraviolet lamps for disinfecting purposes, Journal of the American Medical Association, January 24, 1942, page 298).

The principles which govern the action of ultraviolet on micro-organisms for Vsterilization of liquids are as follows:

1) A definite amount of ultraviolet'energy per time unit is required to kill the infectious agent.

(2,) Ultraviolet, especially in the Schuman range, below 2000 is readily absorbed in clear aqueous solutions and has limited penetration, particularly in turbid fluids containing organic matter.

energy must reach and be absorbed by the infectious agent to exert a lethal effect.

(4) Over-irradiation rapidly destroys the potency and changes chemical structure Ofcompounds.

`Our invention provides the means for producing completely sterile solutions or suspensions containing delicate compounds with minimum denaturation by very rapid exposure of a thin lm to Serial N o. 581,405, produces radiation.

the band designated as 2537 None powerful ultraviolet energy. Ih practicing our inventicn we use a powerful source of ultraviolet,

-which, besides strong emission in the far ultrathe energy in the far ultraviolet.

In the practice of our invention we preferably employ a specially designed ultraviolet permeable irradiation chamber to produce a rapidly flowing, uniform film of a preparation containing microorganisms which can be made as thin as 0.1 mm. Previous methods have employed stationary layers of suspensions in dishes or a column of fluid through a straight or spiral tube, depending on turbulence to expose all parts of the suspension. The exposure of suspensions in dishes as described requires very long periods oftime (fifteen to ninety minutes), so that it not only is commercially impractical because the quantity of liquids is very small, but there is so much over-exposure that a denaturing effect is produced on the proteins and organic substances in the solution. With'the use of suspensions in tubes there were also very long exposures (fteen minutes to four hours) and the method is unreliable and incapable of standardization, so that consistent sterilization is not possible and often the suspension is of no value due to the over-irradiation which occurred. Our combination of an ultraviolet light source of sufficient intensity and a thin, uniform, flowing Atllm of the delicate organic solution enables us to sterilize the contaminating organisms with the least amount. of energy-required for such sterilization, thereby avoiding deteriorating eects from over-irradiation, This procedure can be standardized and is constant, reproducible and reliable. Furthermore, this combination of powerful ultraviolet and rapidly flowing suspension yields large quantities of liquids, sufliciently large for commercial production.

In the practice of our invention we expose a fast-flowing thin film to the radiation of an extreme-low-vapor-pressure mercury are discharge. The output of this extreme-low-vapor-pressure arc can be measured by physical means such as photocells, bolometers, thermopiles and spectographic arrangements. While these measurements are relatively accurate, we. find that biologic means are much more sensitive to determine the output of this extreme low-pressure arc in the extreme ultraviolet and the Schuman range of the spectrum. We have therefore devised a standard test to measure the radiation of the lamp. A tllm 0.1 mm. thick, -containing 1,000,- 000,000 B. coli suspended in physiological saline solution, is exposed to the radiation of the lamp for 1,50 of a second. If all the B. coli are killed, we consider the lamp output adequate. In many instances we expose this suspension for only 50 of a second, which is close to the threshold of sterilization power of a lamp, and the remaining count of living B. coli gives a good indication of the output of bactericidal radiation. If there are proteins in the solution or if spores are present, much longer exposures (several seconds) may be required.

The irradiation cell which we are utilizing in this standard test is described and claimed in our copending application, Serial No. 544,098, iiled July 8, 1944, now abandoned. 'In the test described, tlie extreme-low-pressure arc discharge is operated at an electric input of 500 watts. In the standard test the suspension containing 1,000,000,000 B. coli flows through the irradiation cell at a rate of approximately three liters per hour. Compared with methods of the prior art. this ow is actually many times greater than heretofore achieved, and the procedure of this in venticn can easily be standardized, is constant and reproducible, and is absolutely reliable.

The time required tosterilize or inactivate bacteria or viruses is determined by three factors, first, the resistance of the micro-organism to ultraviolet energy, which varies considerably with the typeof organism to be sterilized or inactivated; second, the count or concentration of bacteria or viruses; and third, the turbidity of the suspension which is to be sterilized.

To cite a specific example, we have been able to render completely sterile human plasma which plasma. and

contained the agent or virus of infectious hepatitis. This is a very valuable procedure and of great importance to public health. Despite great care in thepreparation of human plasma from selected donors, it is possible that this agent might remain in the finished product in spite of the required safety tests, which are inadequate to detect the virus. It is, therefore, good practice to use this invention for the sterilization of all human plasma or serum. As there are no simple means of discovering this virus in plasma, we have adopted a standard routine to sterilize all human serum to make certain that this agent is inactivated. Infectious hepatitis virus is not easily inactivated, and we prefer to expose plasma in a thin nlm, rapidly moving, for a period oi one to two seconds.

In the accompanying drawings we have illustrated an organization of apparatus elements suitable for vuse in practicing the process, the ngure being a side elevation.

In the drawings the lamp illustrated of the type disclosed in the said Oppenheimer pending application comprises a base portion I0 made of high grade optical quartz and having a thickness of substantially 0.5 mm. In a specific embodiment this leg l0 has an inside diameter of l cm. and a length of 16 cm. Section 10 of the lamp is connected to legs Ii which may be of commercial quartz and which are of an over-all length of about 15 cm. Each of these legs is provided with a water jacket I2 having an outlet i3 at its highest point and an inlet H at its lowest point. As shown, the water jacket is provided with an extension i5 covering a portion of a base of the lamp and just beyond the end of the water jacket the commercial quartz forming the leg l i is joined to the high grade quartz forming the base i0 by a suitable seal I6. A pool of mercury l1 is arranged in a bulb or extension i8 which projects beyond the water jacket and which is preferably of a length of about 3 cm. The lamp is connected to terminals I9 of a supply circuit through a switch 20 and an adjustable resistance or reactance coil 2i arranged in series with it.

Plasma is placed in flask 25 and delivered at a v constant rate in close proximity to the leg IIJ of the lamp. In the illustration, the flask 25 is arranged at an elevation to permit controlled flow of the plasma by gravity. The mouth of the flask is closed by a rubber stopper 26. A tube 21 for the inlet of air passes through the stopper and has its end arranged above the level of the plasma i and a length of about Y venous administration after as indicated at 23. The end of the tube outside the flask is also arranged at an eleva'tlon and is filled with a packing 29 of cotton or other suitable material. An outlet tube 3 0 extends through the rubber stopper and is connected to a flexible connector 3l, such as a piece of rubber tubing. The flow of the suspension is controlled by a member 32 which, as shown, may consist. of a capillary glass tube having a restricted portion 33. Any other controlling means may be employed. The other end of this tube is connected to a suitable connector` 34 shown -as :rubber tubing, which is in turn connected to the cell 35 in which the radiation of the suspension takes place. The cell'is also formed of high grade Aoptical quartz readily permeable to wave lengths below 2000 and consists of tubular end portions 36. The intermediate portion 3l' of the cell is flattened forming a passage 38 through it of a thickness of about 0.1 mm. The thickness of the wall of the portion 31 of the cell facing the discharge must be very thin, not over 0.5 mm. The back portion of the cell is thicker to make the cell sturdy. We have` obtained good results with a cell in which the flattened portion 3l is of a width of l cm.

From the cell 35 the, treated suspension ls delivered to asealed ask 39 in which it is collected. As shown, the flask is provided with a rubber stopper 90 having a tube i extending therethrough and the end of this tube is connected to the adjacent end of the cell by a suitable connector, such as a piece of rubber tubing 42. A second tube 43 extends through the stopper 40 and is provided with a packing M of cotton or other suitable material.

Batches of plasma tested in the apparatus and with the lamp above described for the inactivation of the agent of infectious hepatitis has been found to be completely safe and useful for intratwo seconds expos ure. Such irradiation has not resulted in any change of the proteins or decrease oi other delicate substances, such as antibodies contained in plasma.

The present invention is very useful in the sterilization of products of biologic science and industry. Valuable products such as hormone suspensions (liver extract, insulin, adrenalin, pituitrin, thyroxine, etc.) can be sterilized in accordance with this invention without materially impairing their biologic properties. Human plasma and serum can alsobe sterilized without eiect to their chemical constituents, and they remain perfectly safe for intravenous administration. Furthermore, toxoids and antitoxins can be rendered sterile without diminishing their potency. Enzyme solutions, penicillin and other anti-biotics can be treated according to this invention and rendered completely sterile.

We claim:

1. The process ot sterilizing a solution of sus:Y

enslon without impairing potency or altering radiation, in which `the 'thick for a'perlod of not pension without impairing potency or altering chemical or biologic activity which comprises subjecting it in a owing illm not over 1 millimeter A. and zooo .41.15 not 1ess than 1o munwatts per square centimeter and the energy below 2000 is not less than 0.1 milliwatt per square centimeter.

3. The process of sterilizing a solution or suspension without impairing chemical or biologic activity which comprises sub- .iecting it in a ilowing film not over 3 mm. thick for a period of 'not more than 10 seconds to ir- .energy between 2600 and 2000 is not" less than 1 milliwatt per square centimeter and the is not less than 0.1 milliwatt per square centimeter.

4. The process of sterilizing a solution or suspension without impairing potency or altering chemical or biologic activity which comprises subjecting it in a flowing film not over 1 mm. more than 10 seconds to irradiation, in which-the energy between 2600 and 2000 is not less than 1 milliwatt per square centimeter and the energy below 2000 is not less than 0.1 milliwatt per square centimeter.

5. The process of sterilizing a solution or suspension without impairing potency or altering potency or altering chemical or ,biologic activity which comprises chemical or biologic activity which comprisessubjecting itin a owing lm not over 3 mm. thick for a period of not more than 10 seconds to irradiation in'which the energy between 2600 A. and

2000 is not less than 10 milliwatts per square centimeter and the energy. below 2000 A. is not less than 0.1 milliwatt per square centimeter.

2. The process of sterilizing a solution of sussubjecting it to a meters thick for to irradiation in which the energy between 2600 'and 2000 A. is not less than 1 milliwatt per square centimeter and the energy below 2000 A. is not less than 0.1 milliwatt per square centimeter.

` 6. The process of sterilizing a solution or suspension without impairing potency or altering chemical or biologic activity which comprises subjecting it in a flowing illm not over 1 millimeter thick for a period oi' not over 10 seconds. to irradiation in which the energy between 2600 and 2000 isnot less than 10 milliwatts per square centimeter vand the energy below 2000 is not less than 0.1 milliwatt .per square centimeter.

' 7. 'I'he process of sterilizing a solution or suspension without impairing potency or altering chemical or biologic activity which comprises subjecting it in a owing film not over 3 millimeters thick for aV period of not more than 10 seconds to irradiation in which the energy below 2600 Aris' not less than 10 milliwatts per square centimeter.

'i -SIDNEY OLDER LEVINSON. r FRANZ OPPENHEIMER.

REFEttENoEs crrEn UNITED STATES PATENTS owing nlm not over 3 milli- Number Name Date 1,145,140 Henri et al July 6, 1915 2,034,184 Hartman Mar. 17, 1936 2,309,124 Knott Jan. 26. 1943 which the energy between 2600 energy below 2000 a period of not over 1 second 

